Reference electrode having a microfluidic flowing liquid junction

ABSTRACT

A flowing junction reference electrode exhibiting heretofore unattainable potentiometric characteristics is described, comprising a microfluidic liquid junction member that is situated between a reference electrolyte solution and a sample solution. This microfluidic liquid junction member has an array of nanochannels spanning the member and physically connecting the reference electrolyte solution and a sample solution, but while the electrolyte solution flows through the array of nanochannels and into the sample solution at a linear velocity, the sample solution does not substantially enter the array of nanochannels via the mechanisms of diffusion, migration, convection or other known mechanisms. The number of nanochannels in the array is preferably between approximately 10 8  and approximately 10. Also preferably, the nanochannels are substantially straight and are substantially parallel to one another; such an array of nanochannels is herein described as anisotropic. The nanochannels are also preferably coated. The widths of any nanochannels in the array of nanochannels are preferably uniform, in that the width of any nanochannel is substantially equal to the width of any other nanochannels in the array. The nanochannels preferably have widths of greater than approximately 1 nanometer and less than approximately 500 nanometers, and most preferably of 70 nanometers. The electrode may be constructed out of any suitable material, and is preferably is constructed of a polymer, most preferably a polymer selected from the group consisting of polycarbonate and polyimide, and may also preferably be constructed of silicon, glass, or ceramic.

The present application claims priority to, and is acontinuation-in-part of, U.S. patent application Ser. No. 09/590,781,filed Jun. 8, 2000, allowed which in turn claims priority to U.S.Provisional Patent Application Ser. No. 60/138,141, filed Jun. 8, 1999.

This invention was made with United States Government support under SBIRPhase I Grant No. DMI-9960665 awarded by the National ScienceFoundation. The United States Government has certain rights in thisinvention.

PRIORITY CLAIM BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to potentiometric and electrochemical referenceelectrodes and, in particular, to liquid junction structures such as tobe used in electrochemical reference electrodes for electrochemicalmeasurements of solutions. The invention more particularly relates toreference electrodes for use where measurement or control of potentialis desired such as with pH or ISE potentiometric sensors used forlaboratory analysis, for on-line process monitoring, for fieldmeasurements, or in any application where the improved precision orextended useful life of the sensor is desirable.

2. Description of the Related Art

The invention is broadly concerned with reference electrodes, such asthe reference electrode portion of combination electrodes, and thereference portion of all potentiometric devices that employ a referenceelectrode to provide the relatively stable reference potential requiredin various measurements such as electroanalytical measurements,controlled potential coulometry, and polarography, and the like.

Potentiometric measurements are used widely for the determination of pHand the detection of other specific ions in a variety of settings,including chemical processes, environmental monitoring, health care andbio-processes. The accuracy of these measurements depends on the abilityto measure the potential difference between a sensing electrode, whosepotential varies with the analyte concentration in the measured samplesolution, and a reference electrode, which ideally would maintain aconstant potential. The physical interface between the referenceelectrode (typically the electrolyte of the reference electrode) and thesample solution is referred to as the liquid junction. The stability ofthe reference electrode, and consequently the accuracy of potentiometricmeasurements, are dependent on the constancy of the liquid junction andmore particularly, the constancy of the potential across the liquidjunction. However, the liquid junction and more particularly, thepotential across the liquid junction are difficult to control andmaintain at a constant level. Typically, it is the change in theliquid-junction potential that introduces error into the electrochemicalmeasurement and results in the need for frequent sensor systemcalibration.

The errors observed in currently commercially available referenceelectrodes include (1) Transient or kinetic error; such error refers torelatively slow response between measurements, and slow ability to reachequilibrium, typically of five, ten, or fifteen minutes after exposureto extreme solutions. This response is primarily caused by entrapment ofsample solution within the physical junction. Transient errors aretypically a function of the time required to disperse this entrappedlayer of sample solution and obtain a direct interface. The kinetics ofthis error are determined by the duration of prior immersion. The errorsobserved in currently commercially available reference electrodes alsoinclude (2) static error; such error typically refers to persistentoffset after equilibrium is reached. Large static errors are typicallycaused by irreversible entrapment of sample solution deep within thephysical junction structure. The errors observed in currentlycommercially available reference electrodes include (3) stirring error;such error refers to the shift in potential due to or associated withagitation of the sample solution. Stirring error is typically observedwhere there is a rate of agitation or flow of the sample. These errorsexist in potentiometric electrode measurements of sample solutions, buttend to be suppressed in standard buffers where electrode accuracy isbeing checked. Therefore, users may see no reason to disbelieve theerroneous readings obtained in non-standard solutions. See D. P.Brezinski, “Kinetic, Static, and Stirring Errors of Liquid JunctionReference Electrodes”, Analyst 108 (1983) 425-442; see also U.S. Pat.No. 4,495,052. These errors are large enough to be of practicalconsequence. These errors often correspond to relatively largedifference in hydrogen ion (H+) concentration or activity. These errors,including those errors described above, tend to bias the measurementsobserved on pH meters by as much as 0.5 pH unit.

In typical, currently commercially available electroanalyticalmeasurement systems, the interface between the reference electrode'selectrolyte and the sample solution is the liquid junction. The junctionpotential at this sample-reference interface is related to a number offactors; it is an object of every reference electrode design to minimizethe effect of the factors that would cause the liquid junction potentialto drift or to vary in any way over time. Various materials have beenutilized in forming a liquid junction, including porous ceramic rods,porous polymer disks, wood dowls, ground glass sleeves, capillary tubes,agar gels, asbestos fiber bundle, and other porous materials or devices,and the like. These junction structures are, in general, referred to asrestriction devices because their function is to restrict the outwardflow or diffusion of electroyte from the reference electrode. However,one important factor that limits the useful lifetime of a referenceelectrode is that junction structures typically allow the samplesolution to enter the junction structure. This transport of samplesolution into the junction, whether by diffusion, migration, convectionor other mechanism, results in the contamination of the junctionstructure and a resultant undesirable variation in the liquid junctionpotential. Such variation typically necessitates re-calibration of theelectroanalytical measurement system. If this type of contamination ofthe junction continues over time, the junction structure may becomefouled or clogged and develop even larger offset potentials and/orpotentials that chronically drift despite repeated attempts atre-calibration. In addition, sample solution will often transport pastthe junction structure and reach the reference half-cell itself,potentially causing additional adverse reactions.

Currently commercially available reference electrodes, especially thoseused for potentiometric measurements, are typically constructed based onone of two distinct designs. Each of these designs is meant to addressone principle limitation encountered when using reference electrodes formaking potentiometric measurements. However, each of these designs failsto address a distinct principle limitation encountered when usingreference electrodes for making potentiometric measurements.

One design category is often referred to as a flowing junction referenceelectrode. This design provides a stream of reference electrolyteflowing through a porous junction structure or member, in an attempt toprovide a relatively uniform liquid junction potential. While thisdesign is typically effective in providing a liquid junction potentialthat is more uniform over time than those of the alternate design,flowing junction reference electrodes uniformly require the use of largeamounts of electrolyte over relatively short periods of time. Thus,currently commercially available flowing junction reference electrodesrequire frequent maintenance to replenish the supply of this electrolytesolution. Furthermore, while flowing junctions are often designed tominimize this use of electrolyte by restricting the flow of electrolyte,in such flowing junctions designs the flow velocity is often reduced toa velocity that is sufficiently low enough so that the sample solutionenters the liquid junction structure, typically via mass transport(diffusion, migration, or convection). The presence of this samplesolution in the junction structure causes variable junction potentials,loss of calibration, clogging of the junction structure, and, over time,failure of the reference electrode. See U.S. Pat. No. 5,360,529.

The alternative design category is referred to as a non-flowing,diffusion junction reference electrode. This design depends on thesubstantially constant diffusion of electrolyte solution through aminimally porous junction structure to provide a steady liquid junctionpotential. While this design is highly susceptible to mass transport ofthe sample stream into the porous structure, the resulting drift inliquid junction potential may be slow enough to be tolerable in certainindustrial applications. While such electrodes require frequentre-calibration, they do not require replenishment of electrolyte to theextent that flowing liquid junction electrodes do. Furthermore, suchelectrodes do not require systems and associated equipment to feed thereference electrolyte to the electrode, as is the case for typicalliquid flowing junction electrodes.

Both reference electrode designs are in wide use but, based on theirrespective limitations, are typically used in different areas ofapplication. Where precision measurements are more often needed, theflowing liquid junction reference electrode is typically used. Thus theflowing junction design is most commonly used for laboratory referenceelectrodes and clinical analyzers. In the laboratory environment thereference electrolyte may be relatively easily refilled as needed, evenon a relatively frequent basis. Where it is desirable to minimizemaintenance and where precision may be sacrificed to certain degrees,the diffusion junction reference electrode is more often utilized. Thusthe diffusion junction reference electrode is typically used inindustrial potentiometric sensor designs. An industrial sensor that usesa non-flowing, diffusion junction reference will typically requirere-calibration on a more regular basis because of the relatively largeamount of transport of the sample stream into the liquid junctionstructure. It is therefore not unusual for the industrial operator toinstall a new sensor every three months instead of attempting tore-calibrate the old sensor. For this reason, the industrial pH sensorwith a built-in diffusion reference electrode is now a disposable itemin most industrial applications.

In summary, two principal problems with currently commercially availablereference electrodes are the frequent maintenance requirement of theflowing junction design electrodes and the frequent re-calibrationrequirements of the diffusion junction design electrodes. Morespecifically, nearly all flowing junction designs consume large amountsof electrolyte and this electrolyte needs to be replenished on a regularbasis. While there are a few flowing junction designs that require smallamounts of electrolyte, these designs have achieved this by reducing theelectrolyte flow to the point that the level of transport of the samplesolution into the liquid junction structure becomes a limitation. A slowflowing junction reference electrode performs little better than anon-flowing, diffusion junction reference electrode. On the other hand,the non-flowing, diffusion junction electrode requires no electrolytereplenishment but will be subject to slow drift errors due to transportof the sample stream into the liquid junction structure. This drifttypically prevents such reference electrodes from being used forprecision measurements. Frequently, such transport will cause anirreversible instability to develop in the reference electrode that willrender it incapable of being re-calibrated. Because of these inherentshortcomings, sensors employing such reference electrodes are oftendesigned to be thrown away and replaced instead of re-calibrated. As agroup, all non-flowing, diffusion junction reference electrodes have avery short operational life measured in weeks and months and in the bestof circumstances seldom over one to two years

Accordingly, there is a need in the art for an electrode design thatexhibits both the relatively stable potential of currently commerciallyavailable flowing junction designs and the relative lack of the need toreplenish reference electrolyte solution as found in currentlycommercially available non-flowing junction designs. Such a neededdesign would exhibit a relative stable junction potential over prolongedperiods of time, while not exhibiting the various limitations anddrawbacks of currently commercially available flowing junction andon-flowing designs.

SUMMARY OF THE INVENTION

A microfluidic flowing liquid junction (MLJ) member, for use in avariety of potentiometric devices such as reference electrodes orcombination electrodes, is described. This microfluidic flowing liquidjunction comprises nanochannels in a microfluidic structure that createsa substantially invariant liquid junction potential. The microfluidicflowing liquid junctions comprising nanochannels in a microfluidicstructure also preferably exhibit resistances across the junction memberthat are less than approximately 1 megohm. Low volume of flow throughthe array of nanochannels, and high velocities of electrolyte may beemployed to prevent back diffusion of sample solution into the junctionstructure. Prevention of such back diffusion increases the precision anduseful life of a reference electrode having the described junctionmember. The microfluidic liquid flowing junction member is useful toconstruct highly stable, low maintenance, precision electrochemicalsensors, including reference electrodes.

A flowing junction reference electrode exhibiting such heretoforeunattainable characteristics is described structurally as comprising amicrofluidic liquid junction member that is situated between a referenceelectrolyte solution and a sample solution. This microfluidic liquidjunction member has an array of nanochannels spanning the member andphysically connecting the reference electrolyte solution and a samplesolution. The reference electrolyte solution flows through the array ofnanochannels and into the sample solution at a linear velocity, and thesample solution does not substantially enter the array of nanochannels.The sample solution does not substantially enter the array via any masstransfer mechanisms such as diffusion, migration, and convection. Asample solution that enters the array at a rate of less thatapproximately 2×10⁻⁹ moles, and preferably less that approximately1×10⁻⁹ moles per day, should be considered as not substantially enteringthe array. The number of nanochannels in the array is preferably betweenapproximately 10⁸ and approximately 10, more preferably less thanapproximately 10⁶, less than approximately 10⁵, and less thanapproximately 10⁴, and most preferably between approximately 10⁴ andapproximately 100. The number of nanochannels may also be, lesspreferably, between approximately 10 and approximately 1000, includingapproximately 10, approximately 40, approximately 100, approximately200, approximately 400, and approximately 800. Also preferably, thenanochannels are substantially straight and are substantially parallelto one another; such an array of nanochannels is herein described asanisotropic. The nanochannels are also preferably coated, and may becoated with, for example, metals, alloys, hydrophilic materials, orhydrophobic materials. The widths of any nanochannels in the array ofnanochannels are preferably substantially uniform, in that the width ofany nanochannel is substantially equal to the width of any othernanochannels in the array. The nanochannels preferably have widths ofgreater than approximately 1 nanometer and less than approximately 500nanometers, more preferably greater than approximately 10 nanometers andless than approximately 100 nanometers, and most preferably 70nanometers. The electrode may be constructed out of any suitablematerial, and is preferably constructed of a polymer, most preferablythe polymer is selected from the group consisting of polycarbonate andpolyimide, and may also be constructed of other structurally strongpolymers, silicon, glass, or ceramic.

The electrode may also further comprise a pressurized collapsiblebladder, an electro-osmotic pump, or other mechanical pump, or any othermeans for maintaining positive linear flow of the reference electrolytesolution through the array of nanochannels and into the sample solution.The disclosed reference electrode may be used as part of a combinationelectrode along with an appropriate sensing electrode such as a pHelectrode, an ion-selective electrode, a redox electrode, or the like.

A flowing junction reference electrode exhibiting such heretoforeunattainable characteristics may also be described as comprising areference electrolyte solution flowing through a junction member andinto a sample solution; wherein substantially no sample solution entersinto the junction member via mechanisms of mass transfer such asdiffusion, migration, or convection mechanisms. The linear velocity ofthe reference electrolyte solution flowing into the sample solution ispreferably greater than approximately 0.1 cm per second, more preferablygreater than approximately 0.5, and more preferably greater thanapproximately 1.0 cm per second. The volumetric flow rate of thereference electrolyte solution into the sample solution is less thanapproximately 60 μL per hour, and more preferably less thanapproximately 10 μL per hour. The microfluidic flowing liquid junctionreference electrode is capable of having a lifetime of greater than oneyear, and preferably greater than two, three, four, five, or ten years,during which variations of electrolytic potential are less thanapproximately 1 mV per year, and during which less than approximately100 mL of electrolyte flows into the sample solution, and morepreferably less than approximately 50 mL. The resistance across thejunction member electrode is preferably less than approximately 1megohm.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of the specification, are included herein to illustrate certainpreferred embodiments of the invention and, together with the remainderof the written description and claims provided herein, including theDetailed Description of the Preferred Embodiments, serve to explain theprinciples of the invention. The accompanying drawings are not intendedto limit or otherwise define the invention.

FIG. 1 depicts a schematic cross-sectional view of a reference electrodewith means for holding the microfluidic flowing liquid junction in placeat the end of the electrolyte reservoir.

FIG. 2 depicts a detailed schematic cross-sectional view of a means forholding the microfluidic liquid junction structure in place.

FIG. 3 depicts a schematic exploded diametric view of the means forholding the microfluidic liquid junction structure in place.

FIG. 4 depicts a schematic cross-sectional view of certain elements of apreferred microfluidic flowing liquid junction structure and a preferrednanochannel array.

FIG. 5 is an illustrative view representing a single planar, polymermicrofluidic flowing liquid junction structure in which anisotropicnanochannels have been fabricated.

FIG. 6 each depict schematic diametric views illustrating steps in thefabrication of a multiple planar layer polymer junction structure withanisotropic nanochannels and supporting microchannels.

FIG. 7 depicts a detailed schematic cross-section view showing detail ofthe region in which the nanochannels meet a microchannel in a preferredpolymer structure.

FIG. 8 depicts a diametric illustrative view of a microfluidic flowingliquid junction structure having nanochannels and supportingmicrochannels that has been fabricated from one planar element ofsilicone.

FIG. 9 depicts a schematic cross-section view showing the detail ofwhere the nanochannnel meets a microchannel in a silicon microfluidicflowing liquid junction structure.

FIG. 10 depicts diametric views illustrating steps in the fabrication ofa preferred glass microfluidic flowing liquid junction structure frommultiple planar glass elements.

FIG. 11 depicts a schematic cross-sectional view of a glass microfluidicflowing liquid junction structure.

FIG. 12 is a plot of the flux (linear flow) through a nanochannel arrayand the average velocity (v) through a single nanochannel as a functionof the effective radius of the nanochannel.

FIG. 13 is a set of concentration profiles in a liquid junction, plottedas a function of velocity, and as described in Equation (7).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A reference electrode is described that comprises a microfluidic flowingliquid junction having a well-defined junction region, said junctionregion containing a reference electrolyte, wherein said microfluidicliquid junction provides a linear rate of flow of said electrolyte thatis adequate to suppress measurable changes in the electric potential ofthe junction for a period of at least one week, and preferably of longerperiods including at least one month, at least three, six, and ninemonths, and at least one, one and one-half, two and as long ten years.An electrochemical or potentiometric sensor is also described comprisinga pH electrode, an ion-selective electrode, or redox electrode, and areference electrode. The reference electrode comprising means formaintaining a liquid junction potential that remains stable for a periodof at least one week, and preferably for longer periods includingperiods of at least one, two, three, six, or nine months, and at leastone, one-and-one half, and two and as long as ten years.

By using a novel microfluidic junction structure consisting of an arrayof nanochannels, it has been unexpectedly found that heretoforeunattainably stable potentials, low junction potentials, and lowelectrolyte consumption rates for reference electrodes may be produced.These results are preferably attained by using combinations of thenumber of nanochannels and the nanochannel cross-section widths and apositive linear flow velocity for the reference electrolyte through thejunction. The junction structure of the invention may therefore becharacterized by, among other characteristics, (1) high electrolytevelocities to suppress transient, static, and stirring errors; (2)substantially constant junction potentials; (3) substantially constantpotential despite the existence of flow rate and flow velocityfluctuations within the junction; (4) small junction potentials; (5) lowjunction resistance; and/or (6) extremely low consumption ofelectrolyte.

It is therefore one object of the invention to provide a referenceelectrode with a flowing liquid junction structure that will maintain aheretofore unavailable relatively constant, invariant, and fixedjunction potential, such potential being maintained for extended periodsof time, including periods of one month to up to one, two, three, andeven ten years, without the need to replenish the reference electrolyte.

Another object of the invention is to provide a flowing liquid junctionthat functions for relatively prolonged periods of time on a relativelysmall amount of electrolyte and provides a substantially constant liquidjunction potential that is substantially free of transient errors,static errors, and stirring errors.

It is another object of the invention to provide a reference electrodethat will have minimal transient, static, or stirring errors in samplesolutions of extreme pH, solutions having relatively high concentrationsof highly charged ions, and/or solutions having low ionic strength.

Another object of the invention is to provide a flowing liquid junctionthat is neither a “leak path” nor a “restricted diffusion” junction. Inthis flowing liquid junction of the invention, there is hydrodynamictransport across the junction structure or member into the samplesolution. This hydrodynamic transport is preferably at a velocitysufficiently high to effectively counter back diffusion of the samplesolution into the nanochannels of the junction. Prevention of this backdiffusion contributes to the junction potential remaining stable andfree of transient, static, and stirring errors for prolonged periods oftime of one month to up to one, two, three, and even ten years.

Another object of the invention is to provide a flowing junctionstructure that provides a constant liquid junction potential over abroad range of electrolyte flow velocities. The liquid junctionstructure provides a constant potential that is relatively andsubstantially free of fluctuations even as the electrolyte velocityvaries within various velocity ranges.

It is another object of the invention to provide a flowing junctionreference electrode that functions for relatively long periods of timewithout the need for replenishment of the reference electrolyte or theassociated maintenance. The reference electrode according to theinvention may thus function for times of 1, 10, 20, 30, 40, 50, 60, 70,80, 90 or even 100 years while using less than 100 mL of electrolyte.

It is another object of the invention to provide a flowing junctionreference electrode that uses such small amounts of electrolyte that theelectrode will consume as little as 1 mL of electrolyte per year.Certain preferred embodiments of the invention allow the referenceelectrode to function for as long as 10, 20, 30, 40, 50, or as long as100 years on only 100 mL of electrolyte, or in other embodiments, lessthan a mL per week, a mL per month, or a mL per six months.

It is another object of the invention to provide a junction structurethat consists of an array of nanochannels that provide low electrolyticresistance. While each separate nanochannel is high in electrolyticresistance, the entire array of nanochannels provides a junctionstructure having an electrolytic resistance that is relatively low.

It is another object of the invention to provide a pressurized array ofnanochannels that achieves a linear velocity of electrolyte necessary tosubstantially and effectively counter back diffusion of the samplestream into the junction and thus avoid transient, static, and stirringerrors.

It is another object of the invention to provide a pressurized array ofnanochannels that achieves a linear velocity of electrolyte necessary tosubstantially and effectively reduce fouling and blockage by gas bubblesor particulate matter.

It is another object of the invention to provide a pressure differentialacross the array of nanochannels, through which reference electrolyteflows. The volume of each typical nanochannel in the array issufficiently small that high electrolyte velocity can be achieved forprolonged periods of time with the use of extremely small volumes ofelectrolyte. These prolonged periods of time can be as long as years andeven decades.

It is another object of the invention to provide a reference junctionstructure sufficiently robust to function in a process industrialenvironment and sufficiently small to be incorporated as a basicbuilding block into portable microfluidic module-based analyticaldevices.

It is another object of the invention to provide a liquid junctionstructure that can be miniaturized for compatibility and integrationinto microfluidic devices, such as for example hand-held analyticdevices for use in remote locations, and portable analytic devices foruse in field stations, battlefield hospitals, emergency stations or thelike.

Another object of the invention is to produce a reference junctionstructure with a nanochannel array that may be manufactured with planarfabrication techniques so that the reference junction structure may bebatch produced as an integral component of the various microfluidicstructures and devices.

Another object of the invention is to provide an substantially invariantliquid junction structure that can be fully integrated into mesoscaleand microscale microfluidic devices.

It is another object of this invention to provide a liquid junctionstructure that can be miniaturized for compatibility and integrationinto microfluidic devices. A further, related object of this inventionis to provide a liquid junction structure, the manufacture of which maybe achieved through the use of current microfabrication techniques.

A device need not attain even one of these objectives to be within thescope of the invention.

General Discussion of the Uses and Design of Reference Electrodes

The microfluidic flowing liquid junctions and reference electrodesincorporating such microfluidic flowing liquid junctions, as disclosedherein, expand the use of electrochemical monitoring to remote and/orhazardous sites, and to in-line process conditions. Their use willresult in lower cost and improved efficiency of monitoring andcontrolling chemical and biological industrial processes. A referenceelectrode that extends the useful lifetime of a sensor and maintains acalibration for prolonged periods dramatically reduces maintenancerequirements, increases efficiency, and decreases costs.

Reference electrodes are most typically used for example in thefollowing way: In the measurements of ion concentration of solutions, areference electrode is commonly employed in conjunction with a sensingelectrode, such as a glass pH electrode, with both electrodes immersedin the test solution. The potential difference between the twoelectrodes is a function of the concentration of the specific ion insolution. A typical example is the conventional pH meter and pHelectrode pair used for measuring hydrogen ion concentrations ofsolutions.

Reference electrodes are also frequently used in conjunction with anion-sensing electrode such as a pH electrode or a redox electrode,either separately or in combination, to measure the activity (which is afunction of the concentration) of a given ion in a sample solution. Thetwo electrodes, for example, the reference electrode and theion-selective electrode or the reference electrode and the redoxelectrode, both of which are immersed in the sample solution, typicallyare connected to a means of measuring the potential difference betweenthe two electrodes, for example, an electrometer. The referenceelectrode is expected to provide a constant electromotive force orpotential against which the potential of the ion-selective electrode iscompared. The latter potential consists of a constant component from theelectrochemical half-cell of the ion-selective electrode and a variablecomponent which is the potential across the sensing membrane and whichis dependent upon the activity (concentration) of the ion beingmeasured. The variable component, then, is readily correlated with ionactivity (concentration) by known means. To give accurate results, thepotential of the reference electrode should not change with thecomposition of the sample.

When used in such applications, reference electrodes are meant toestablish a relatively constant or stable potential, which in an idealsituation is independent of the composition of the liquid sample, but inpractice varies with the liquid junction potential. The liquid junctionpotential is the potential difference, created across the interfacebetween the sample solution and the reference electrolyte. Thisinterface is typically present at the junction member. The junctionpotential will vary with varying dilution and varying ion compositionbetween sample and electrolyte. These variations affect the measuredresults and they will become imprecise or misleading over time.

A reference electrode is typically comprised of an internal half-cellsupported in a tube containing a salt solution, the tube of saltsolution being known as a salt bridge. The salt bridge solution is astrong equitransferent salt solution such as potassium chloride orpotassium nitrate. Electrical connection between the salt solution andthe sample or test solution is made by liquid flow through a suitablyformed aperture or passage in a tube, generally referred to as theliquid junction structure or the leak structure. Sometimes the entireunit consisting of the internal half-cell structure, the tube, the saltsolution, and the liquid junction structure is referred to as ahalf-cell; however, for the present specification, the entire unit willbe referred to as a reference electrode.

Definitions

As used herein, the term “nanostructures” refers to assemblies that havedimensions in the range of approximately 1 to approximately 500 nm.Accordingly, “nanochannels” refer to channels having widths ofapproximately 1 to approximately 500 nm.

As used herein, the terms “mass transfer” and “mass transport” eachrefer to mechanisms for the flow of mass including diffusion, migration,and convection.

As used herein, the phrase “the sample solution does not substantiallyenter the array of nanochannels” refers to the substantial absence ofback diffusion of the sample solution into the nanochannels of thejunction where such back diffusion would measurably alter the potentialof the reference electrode.

As used herein, the term “microfluidic” refers to a structure or devicehaving channels or chambers which are generally fabricated at the micronor submicron scale. Such structures and devices preferably have at leastone cross-sectional dimension in the range of about 10 nm to about 500microns. Techniques commonly associated with the semiconductorelectronics industry, such as photolithography, wet chemical etching,etc, are typically used in the fabrication of microfluidic structures.Such structures may be batch fabricated in, for example, silicon,polymers (including plastics), ceramic, glass, and quartz, using planarintegrated circuit fabrication techniques.

As used herein, “fluid mechanics” refers to the study of motion andcontrol of fluids. Micromachined fluid components offer the potential ofrevolutionizing applications where precise control of fluid flow is anecessity. Microfluidic systems comprising nozzles, pumps, channels,reservoirs, mixers, oscillators, and valves have been used in a varietyof applications including drug dispensation, ink-jet printing, andgeneral transport of liquids, gasses, and liquid/gas mixtures. Theadvantages of these devices include lower cost, enhancement ofanalytical performance, and lower consumption of reagents.

As used herein, the term “half-cell electrode” means the solid-phase,electron-conducting contact with the half-cell electrolyte, at whichcontact the half-cell oxidation-reduction reaction occurs whichestablishes the stable potential between the half-cell electrolyte andthe contact. See, e.g., U.S. Pat. No. 4,495,052.

As used herein, the term “electrochemical” refers to any use and/orsensor that exploits electrochemistry; and includes within it the term“potentiometric.”

Manufacture of the Invention

Microfabrication of electrochemical sensors using integrated circuit(IC) technology has been challenged by the failure to incorporate a truereference electrode into the structure. See Mark Madou, “Fundamentals ofMicrofabrication,” 1997, CRC Press, pg. 469. There is great potentialfor developing simple devices that are inexpensive, easy to fabricate,disposable, and highly sensitive. These devices can prove to be simpleminiaturized diagnostic tools for various state-of-health indicators.

Back diffusion of sample solution into the physical junction generates ajunction potential that not only shifts the calibration (generatingstatic error) but may also cause the sensor signal to drift at anymeasurement point (generating transient error). Such back diffusiongreatly increases the frequency of calibration required to obtainprecise data from the electrochemical sensor. This increases the cost ofownership and places limits on the amount of time that such a device canfunction unattended. This is especially a problem for remote sensingdevices that monitor water chemistry in lakes and streams and have aneed to operate for extended periods of time without maintenance orrecalibration.

Most attempts to minimize back diffusion require a flowing junctionstructure that needs large amounts of electrolyte and periodic refillingof the electrolyte reservoir and other associated maintenance. This addsto the operational complexity of the sensor device and increases thecost of ownership by requiring scheduled maintenance by a technician.This is especially a problem with remote environmental measuring devicesthat are deployed to monitor lake and stream water chemistry.

Volumetric flow rate and electrolyte consumption are typicallycompromised one for the other; decreasing one parameter increases theother. As stated above, it is therefore an object of this invention toprovide a reference structure that prevents back diffusion whilesignificantly increasing the linear velocity of the electrolyte flowingthrough the nanochannel array and minimizing volumetric flow rate. Thisvelocity suppresses back diffusion of the sample into the referencestructure and enables the reference electrode to be operated forextended periods of time without the need for recalibration.

Embodiments of the invention provide a junction structure that employsan array of nanochannels in a microfluidic structure to achieve a highelectrolyte velocity while at the same time utilizing very lowvolumetric flow rates and using only sparingly small amounts ofelectrolyte solution. The microfluidic structure with its array ofnanochannels can operate from 1 to 100 years on 100 mL of electrolyte.Alternatively a single milliliter of electrolyte could enable a small,disposable measurement device to operate with laboratory precision from2 weeks to a year in harsh environments such as battlefield fieldhospitals.

Embodiments of the present invention substantially mitigate these longstanding problems of reference junction stability and electrolyteconsumption. With the embodiments of the present invention,potentiometric sensors systems can function for extended periods of timewithout the need for recalibration or electrolyte replenishment.

Embodiments of the present invention provide a microfluidic referencejunction structure that enables precise potentiometric measurements tobe made with devices and systems that operate remotely and withoutmaintenance for long periods of time.

This reference structure can be miniaturized for compatibility andintegration into microfluidic devices. Such miniaturization can besubject to performance and stability trade off's with existing junctionstructures. The microfluidic flowing liquid junction described hereinachieves its superior performance because of its nanoscale structure. Itis already small enough to be included as a subcomponent in a microscaledevice such as a disposable microfluidic chip, disk, or block. Yet thesame microfluidic flowing liquid junction structure is robust enough tobe readily utilized as the liquid junction of a macroscale industrialin-line sensor assembly or a mesoscale analytical handheld device.

A reference electrode with an substantially invariant liquid junctionpotential using an innovative combination of microfluidic andnanotechnology is described. The variability of the liquid junctionpotential is a significant factor in the accuracy of potentiometricmeasurements. Removing this variable will result in potentiometricmeasurements with improved stability, precision and reproducibility. Areference electrode with an substantially invariant liquid junction iscapable of sustaining a single calibration for prolonged periods.Reducing the calibration and maintenance will diminish the cost andenhance the ability to monitor remote and hazardous sites.

The reference electrode described herein preferably uses microfluidicconcepts to incorporate a nanochannel array for the liquid junctionstructure. This microfluidic flowing liquid junction preferablymaintains a constant potential reproducible to ±0.5 mV (˜0.01 pH unit)and preferably has a life in excess of one year. An important factor isthe stability of the liquid junction. In an electroanalytical system theinterface between the reference electrolyte and the sample solutionconstitutes the liquid junction. Unless these two solutions have thesame initial composition, the system will not be at equilibrium. Thoughthe liquid junction region is not at equilibrium, if it has acomposition that is effectively constant, then the reversible transferof charge through the region can be considered. See Bard, A. J.;Faulkner, L. R. Electrochemical Methods; John Wiley & Sons: New York,1980; pp 61-64. Providing an adequate outward flow of junctionelectrolyte serves to suppress changes in the junction potential. SeeBrezinski, D. P. The Analyst 1983, 108, 425. Maintaining a constantcomposition, and narrow, well-defined liquid junction region, thereforeprotects the reference electrode's liquid-junction potential stability.The system uses small volumes of electrolyte to make it a practicaldevice for operation for one year or more with a reduced level or nomaintenance.

Factors that affect the liquid junction potential include temperature,ionic strength, and transport of ionic and molecular species across thereference structure. The most stable and reproducible referenceelectrodes use a flowing-liquid junction. The continuous flow ofelectrolyte maintains a constant rate of ion transport across theinterface. In addition, the constant flow of electrolyte also preventsback diffusion of the sample into the reference electrolyte. However, aconventional flowing junction can use large quantities of electrolyteand require substantial maintenance, which is impractical in mostindustrial applications.

The microfluidic flowing liquid junction may be comprised of nanochannelarrays in a structure that results from recent developments inmicrofluidic and nanotechnology. This technology makes it possible togenerate sufficient electrolyte flow through the liquid junction toeliminate contamination of the junction structure, yet use only minimalquantities of electrolyte. The microfluidic flowing liquid junctionpreferably maintains a constant potential for an extended duration oftime, and preferably limits the volume of electrolyte to a volume rateof flow of less than 50 mL per year (6 μL per hour). This allows forreference electrodes, and consequently potentiometric or electrochemicalsensors that require neither maintenance nor recalibration for periodsof preferably at least one week, two weeks, one months, six months, orone year.

The feasibility of using the microfluidic flowing liquid junction, maybe demonstrated by: (i) determining the electrolytic resistance acrossthe nanochannel arrays; (ii) characterizing the flow of electrolytethrough nanochannels as a function of applied pressure, nanochannelmaterial, and nanochannel dimension, (iii) determining the requiredelectrolyte velocity through a nanochannel to eliminate back diffusionof the sample solution into the reference electrode, and (iv) building alaboratory reference electrode and demonstrate a stable referencepotential using a microfluidic flowing liquid junction.

Furthermore, the microfluidic flowing liquid junction may be furtheroptimized as follows: (i) optimizing the electrolyte velocity,nanochannel materials and dimensions, (ii) developing appropriatepumping mechanisms and designs.

The following description of the present invention is divided into twosections. The first section is a technical discussion of themicrofluidic flowing liquid junction and its use in a referenceelectrode, including theoretical and conceptual discussions of theliquid junction and its potential, transport through microchannels, andthe utility of nanochannel arrays. The second section lists anddescribes methods to achieve various tasks, including a discussion ofthe tests and experiments used to demonstrate the functionality of amicrofluidic flowing liquid junction for a reference electrode.

A Reference Electrode Having a Microfluidic Flowing Liquid Junction

Prototypes with a microfluidic flowing liquid junction are assembled inthe following manner. The preferred junction has a modular design foreasy exchange of different nanochannel arrays. Six electrodes areconstructed so that simultaneous measurements can be made. Thenanochannel array is sandwiched between two silicon rubber gaskets (idapproximately 1 mm). The gaskets can be compressed and sealed to theelectrode body. The electrode electrode allows variable internalpressures. The reference electrolyte is forced to flow by applying apneumatic pressure on the reference reservoir. The differential pressureis limited to 40 psi or to 100 psi. The reference reservoir containsapproximately 50 mL of 4.0 M KCl, and uses a Ag/AgCl referenceelectrode.

Determination of the Electrolytic Resistance of the Nanochannel Array

The electrolytic resistance of the nanochannel arrays is measured by ACimpedance. A Solartron AC impedance system is available. The nanochannelarray is clamped between the two halves of a U-tube permeation cell.Both half-cells are filled with 4.0 M KCl. The working and referenceelectrodes are placed in one half-cell (on one side of the array); thecounter electrode is placed in the other half-cell (on the other side ofthe array). The impedance at high frequencies (e.g., 50 kHz to 100 kHz)is real and corresponds to the solution resistance. In thisconfiguration, the solution resistance has three components; theresistance in one half-cell, the resistance in the second half-cell, andthe resistance of the nanochannel array. The resistances of thehalf-cells are negligibly small relative to the nanochannel arrayresistance. This may be verified by repeating the same experimentwithout the array. If necessary, the measured solution resistance fromthis experiment will be subtracted from the measured resistance when thenanochannel array is in place. The measured resistances may be comparedto calculated values obtained using eq. (3) below.

Characterizing the Electrolyte Volumetric Flow Rate and Linear Velocity

The flow rate and velocity of the reference electrolyte through thenanochannel arrays are determined as a function of applied pressure,nanochannel dimension, and nanochannel material. The applieddifferential pressure may be varied from 0 (diffusion) to 40 psig. Theflow rate may be measured by placing the junction in 50 mL of ultra-purewater and measuring its transient conductivity. The experimentallydetermined flow rates may be compared to the predicted flow rates,calculated using eq. (2) below. The linear velocity may be calculatedbased on the pore density and dimensions of the nanochannel array.

The effect of charged nanochannel walls on the transport of thereference electrolyte may also be studied. Chloride ions readily adsorbon gold surfaces, thus, the Au nanochannels may have a net negativecharge. In this situation, the nanochannels are cation permselective.However, if the nanochannels are pretreated with propanethiol they havean inert, neutral coating, and chloride ions do not adsorb. To determinewhat effect charged walls may have on the transport, flow rates throughAu nanotubles with negatively charged and neutral walls may be compared.This comparison provides useful information on the transport mechanismof permselectivity with pressure driven flow through nano-sized pores.

Measuring Back Diffusion as a Function of Linear Velocity of ElectrolyteSolution

Back diffusion as a function of velocity may be measured using acustom-designed pressure cell. Such a cell consists of feed and permeanthalf-cells. The feed half-cell will contain the 4.0 M KCl. The permeanthalf-cell may be a dilute aqueous solution of a strongly absorbing dyemolecule (e.g., Rhodamine B). The back diffusion of the dye from thepermeant into the feed may be measured spectrophotometrically as afunction of applied pressure. The rate of back diffusion may be measuredby following the time-course of the dye appearance into the feed cell.The velocity of solution flow from the feed to the permeant may bemeasured by monitoring the conductivity of the permeant (due totransport of KCl from the feed) as a function of time. In this way, theminimum solution velocity (feed to permeant) required to eliminate backdiffusion of dye (permeant to feed) into the reference electrode chamberwill be determined.

Comparing Microfluidic Flowing Liquid Junctions to Standard ReferenceJunctions

A reference electrode having an microfluidic flowing liquid junction maybe compared to traditional reference junctions to determine its relativepotential and utility for reference electrodes. A reference electrodewith a microfluidic flowing liquid junction may be used for pHmeasurements, and its response may be compared with different referenceelectrodes. The overall stability and performance of a referenceelectrode is determined from (i) transient error, (ii) static errors,and (iii) stirring errors.

First, when an electrode is transferred from one solution to another, ifany of the first solution is retained within the liquid junction, themeasured potential should have a contribution from the originalsolution. This is referred to as a memory effect, or transient error.Notwithstanding any permanent contamination, the liquid junction can berenewed by the continuous outflow of reference electrolyte. Memoryeffects, transient errors, may be determined by measuring the timerequired to achieve a steady potential response. The response times ofthe microfluidic flowing liquid junction may be compared with typicalflowing, and diffusion-style reference junctions.

Second, stirring the sample solution can change the measured pH.Stirring can effect the potential measurement in at least two ways.Streaming potentials can build-up from convection of the samplesolution. This becomes evident when the ionic concentration of thereference electrolyte differs from the sample, especially in low ionicstrength sample solutions. In addition to streaming potentials, stirredsample solutions can increase contamination of the liquid junction.

The effect of pressure in the sample solution may be measured up to 40psig, in or alternatively to 50, 60, 70, 80, 90, and 100 psig. Thepotential dependence of the microfluidic flowing liquid junction ontemperature may then be determined.

Performance the Microfluidic Flowing Liquid Junction Over Extended Times

The microfluidic flowing liquid junction references may be placed instandard pH buffers for extended periods. The long-term testing may alsobe conducted in different media, including wastewater and soils. Themicrofluidic flowing liquid junction preferably retains its calibrationto within 0.5 mV over a 24-hour period in adverse test conditions.However, a microfluidic flowing liquid junction preferably sustains asingle calibration for even greater prolonged periods of time.

Certain Preferred Aspects of the Microfluidic Flowing Liquid Junction

Certain preferred aspects of the invention, many of which are furtherelucidated through the specific examples described herein and many ofwhich may be observed in the various embodiments of the invention, areas follows:

According to a preferred aspect of the invention, there is provided anarray of electrolyte flow channels in the junction member. As shownherein, an array, as opposed to a single channel lowers the overalljunction resistance while minimizing electrolyte consumption. Eachchannel can be very high in resistance while the sum resistance of allthe channels of an array will be several orders of magnitude lower inresistance. Without an array, or plurality, of channels the junctionstructure resistance would typically be too high for practical use.

According to another preferred aspect of the invention, there isprovided an array of nanochannels in the junction member. Channelshaving internal diameters in the lower end of the nanometer range (forexample, less than approximately 100 nm or approximately 70 nm) permitachieving the preferred elevated electrolyte solution linear velocityand the substantially constant liquid junction potential while consumingonly relative small amounts of electrolyte solution. The array ofnanochannels may also comprise approximately 10³, 10⁴, 10⁵, or 10⁷nanochannels. The volume rate of flow is preferably less thanapproximately 50 mL per month, and may also be less than approximately 2liters, 1 liter, 500 ml, 300 ml, 250 ml, 200 ml, 150 ml, or 100 mL permonth, and more preferably less than approximately 50 mL per year, andmay also be less than approximately 2 liters, 1 liter, 500 ml, 300 ml,250 ml, 200 ml, 150 ml, or 100 mL per year. Also, the linear flow rate,dependent on the radii or effective width of the nanochannels employed,is preferably greater than approximately 0.1 cm per second, and,depending on the radii or effective width of the nanochannels, may begreater than 0.0001 cm, 0.001 cm, and 0.01 cm per second.

According to another preferred aspect of the invention, there areprovided aniostropic channels in the junction member. Such channels aresubstantially straight and parallel to one another, and with uniformpore size provide substantially uniform distribution of flow throughsubstantially all channels. Such channels may preferably be preparedaccording to the “template synthesis” method described herein and inHulteen, J. C.; Martin, C. R. J. Mater. Chem. 1997, 7, 1075.

According to another preferred aspect of the invention, there areprovided channels having internal diameters of less than approximately100 nanometers or approximately 70, 50, 40, or 30 nanometers. Channelsof these dimensions enable obtaining the preferred combination ofelectrolyte flow velocity, minimum electrolyte consumption, and arrayresistance.

According to another preferred aspect of the invention, there areprovided channel lengths greater than approximately 100 nanometers andless than approximately ten microns. Channels at this dimension range(or smaller) also enable obtaining the preferred combination ofelectrolyte flow velocity, minimum electrolyte consumption, and arrayresistance.

According to another preferred aspect of the invention, there isprovided a number of channels less than approximately one-hundredmillion (10⁸). Arrays with fewer than this number of channels enable adesirable combination of electrolyte flow velocity, minimum electrolyteconsumption, and array resistance.

According to another preferred aspect of the invention, there isprovided a driven flow with high electrolyte velocity greater thanapproximately 0.1 cm/sec. Flow velocity is a factor in determining thepreferred flow rate of electrolyte through the junction. Velocities atthis rate or higher are necessary to substantially prevent penetrationof each nanochannel by sample solution. Contrary to the commonly usedtechnique of restricting the flow rate (volume and velocity) to minimizeelectrolyte consumption, preferred embodiments of the present inventiongreatly accelerate velocity in a nanochannel structure while usingrelatively small amounts of electrolyte.

According to another preferred aspect of the invention, there isprovided reduced volumetric consumption of electrolyte. Flowing junctiondesigns traditionally use relatively large quantities of electrolyte andneed frequent replenishment and associated maintenance. The designparameters of this reference junction provide superior electrolytevelocity with vastly reduced flow volume of reference electrolyte. Forexample, as little as one mL per year, is consumed under standardoperating conditions. Preferred embodiments of the invention providejunction designs that can function for prolonged periods of time withoutthe need for electrolyte replenishment and minimal contamination of thesample. Certain embodiments of this invention can, for example, operateup to 90 years with only 100 mL of electrolyte.

According to another preferred aspect of the invention, there isprovided a low junction resistance: having a resistance across junctionof less than approximately 1000 kiloohms (1 megohms). The microfluidicflowing liquid junction electrode is shown to achieve high velocity andlow volume electrolyte use without sacrificing junction resistance.

According to another preferred aspect of the invention, there isprovided a junction that maintains a stable junction potential over awide range of junction flow rates and flow velocities. Unexpectedly, thenovel junction does not generate a different internal potential atdifferent flow rates or flow velocities. Such a result is contrary toprior teachings. This unexpected property alleviates the need formaintaining a constant flow rate or velocity. Importantly, in apressurized driven device, the flow rate will decrease as theelectrolyte is depleted. Contrary to teachings and expectations, thejunction potential has remained constant over a wide range of pressuresand flow rates. For this reason, the electrolyte solution may be held ina flexible, pressurized collapsible bladder.

According to another preferred aspect of the invention, there isprovided a reference electrode that may readily be integrated with anyknown variety of sensing electrode to make a combination sensor.

According to another preferred aspect of the invention, there isprovided a combination sensor that may employ a battery poweredcompensating circuit. The circuit is designed to substantially null theinherent offset in the sensor and maximize the slope of the sensorresponse between two standards.

According to another aspect of the invention, it becomes unnecessary tomaintain a constant pressure across the junction. The pressure may varyfrom high as 40 psig to as low as 10 psig and maintain substantially noerror.

According to another aspect of the invention, various mechanisms may beused to maintain desired flow of electrolyte solution through thejunction member. For example, a pneumatic driven flow or pump, such as acollapsible bladder, or electro-osmotic flow or pump orelectro-hydrodynamic flow or pump may also be used. Also, for example, amechanical pump or flow such as a piston-driven pump or flow may beused, or a spring-driven piston pump or flow, or a piezo-electric flowor pump or an electro-hydrodynamic flow or pumps may be used. Such pumpsare well known in the art and are described by Marc Madou in“Fundamentals of Microfabrication”, 1997, CRC Press, pg. 431-433.

According to another aspect of the invention, the inner walls of themicrofluidic flowing liquid junction may be physically or chemicallymodified to alter the flow of electrolyte. For example, the inside wallsof the structure may be coated with substances to enhance flow ofelectrolyte. Also, for example, the inside walls of the structure may beplated with metals such as gold, platinum, or palladium or anothernon-reactive metals or alloys or combinations thereof to increasefunctionality and to effect additional functionality or performancegains. Also, for example, the walls may be made hydrophilic by theaddition of for example, a hydrophilic polymer such as a polythiol orpolyvinylpyrolidone (PVP) or a hydrophobic material. Also a surfactantmay be added to the electrolyte to alter the flow of electrolyte throughthe nanochannels, especially of the smaller nanometer structures.

EXAMPLES

The microfluidic flowing liquid junction and associated electrodes ofthe invention are described in terms of several embodiments. Theseembodiments are preferred and comprise microfluidic liquid junctionstructures with nanochannel arrays fabricated from a variety of specificmaterials. Each preferred structure may be fabricated, according totechniques known in the art, into a thin wafer or membrane, preferablyround, that can be mounted onto the end of a reference electrodestructure. Each junction structure permits electrolyte flow through ananochannel array from the internal electrolyte reservoir of thereference electrode into the sample solution.

FIG. 1 depicts a representative diagram of an exemplary potentiometricreference electrode 100 with a microfluidic liquid junction structure102 according to the present invention. The reference electrode 100comprises of a chamber 114 that has a seal 120 on one end and acompression means 122 for sealing the junction structure 102 in place atthe other end. The reference electrode 100 includes an electrochemicalhalf-cell 108, an electrical conductor 118, and a reservoir of referenceelectrolyte solution 110. The electrolyte reservoir 110 is contained ina flexible elastomer reservoir bag 112 that separates the electrolytereservoir 110 from the compressed gas 116 that fills the rest of thechamber 114. The compressed gas 116 compresses the reservoir bag and theelectrolyte therein and by this means drives the electrolyte 110 throughthe aperture 130 and into and through the microfluidic flowing liquidjunction member and out the orifice 132 and into the sample stream (notshown). In this manner the reference electrode 100 shown in FIG. 1utilizes the microfluidic flowing liquid junction structure 102 to makeelectrolytic contact between the internal electrochemical half-cell 108and the sample solution (not shown).

FIG. 2 depicts a cross-sectional view of the compression means 122 thatseals the microfluidic liquid junction 102 structure onto the end of thereference electrode chamber 114. The threaded retainer ring 124compresses the microfluidic liquid junction structure 102 against theo-ring 126 and the gasket 128 and thereby seals it into the end of thereference electrode chamber 114. The pressurized electrolyte 110 ispushed through aperture 130 and into and through the microfluidicsliquid junction structure 102 then out of the orifice 132 and into thesample stream (not shown).

FIG. 3 depicts an exploded diametric view of the compression means 122.In this example of the embodiment the microfluidic liquid junctionstructure 102 is a round planar element.

FIG. 4 depicts a schematic cross-section of the microfluidic liquidjunction structure 102 in its most elementary form, a single planarelement. As shown, the microfluidic liquid junction structure 102 isfabricated in a planar substrate 104. Suitable substrate materials aregenerally selected based upon cost, ease of fabrication, dimensionalstability, mechanical strength, and compatibility with the conditionspresent in the particular environment that the structure will beoperating in. Such conditions can include extremes of pH, temperature,ionic concentration, and presence of organic solvents. Useful substratematerials include glass, quartz, ceramic, silicone, polysilicone, aswell as polymeric materials such as polycarbonate, polyimide, and otherplastics typically utilized in microfabrication techniques.

The junction structure 102 includes a multitude of nanochannels 106fabricated through the substrate 104 and generally perpendicular to theplanar axis of the substrate 104. These nanochannels typically have verysmall cross-section dimensions, preferably in the range from about 1 nmto 500 nm. It is this small, nanometer scale, cross-sectional width ofthe nanochannels 106 that gives them their name. For the particularpreferred embodiments, nanochannels 106 that have cross section widthsof about 10 nm to 100 nm and lengths of about 0.5 μm to 200, 300, or asgreat as 500 μm will work most effectively, although deviations fromthese dimensions are within the scope of the invention.

The multitude of nanochannnels 106 present in the microfluidic liquidjunction structure 102 are referred to collectively as the array 105.The size of the array 105 is characterized by the number of nanochannels106 present in the structure 102. The number of nanochannels can varyfrom 10 to 100,000,000. More generally, the number of nanochannels canbe selected from any whole number less than 10⁹, and can be as low asdesired, provided that the effects of the plugging of one or morechannel will not substantially and adversely effect the performance ofthe MLJ. For these particular embodiments discussed below, an array 105with a number of nanochannels 106 between 1000 and 1,000,000 will workmost effectively, though deviations from these numbers are within thescope of the invention, as noted above.

The array 105 of nanochannels 106 is a common element in all depictedembodiments of the invention and the operational characteristics of aparticular array may be predicted by specifying only three parameters ofthe array 105: (1) the cross-sectional width of the nanochannel 106, (2)the length of the nanochannel 106, (3) and the number of nanochannels106 present in the array 105. Table 1 provides ranges expressed inapproximate values preferred ranges, for these three parameters.

TABLE 1 Representative Approximate Ranges for Nanochannel ArrayParameters Individual Nanochannel 106 Cross-sectional Width Range: 1 nmto 900 nm Preferable ranges: 10 nm to 500 nm; 40 nm to 100 nm; 70 nmIndividual Nanochannel 106 Length Range: 0.5 μm to 500 μm Preferableranges: 0.5 μm to 100 μm; 6 to 10 μm Number of Individual Nanochannels106 in Array 105 Range: 10 to 100,000,000 Preferable ranges: 1000 to1,000,000

Manufacturing of the array 105 of nanochannels 106 and other micro- andnanoscale elements and features into the substrate 104 may be carriedout by any number of microfabrication techniques that are well known inthe art. For example, photolithographic techniques may be employed infabricating glass, quartz, ceramic, silicone, polysilicone, or “plastic”polymeric substrates with methods well known in the semiconductormanufacturing industries. Photolithographic masking, plasma or wetetching and other semiconductor processing technologies definemicroscale and nanoscale elements in and through the substrate and onthe substrate's surfaces. Alternatively, micromachining methods, such aslaser drilling, micromilling, microgrinding, and the like may beemployed. Similarly, for polymeric substrates, such as plastics, wellknown manufacturing techniques may be used. These techniques includecharged particle bombardment and subsequent wet etching of nanoscale andmicroscale channels through polymeric substrates. Additional techniquesinclude injection molding techniques or stamp molding methods wherelarge numbers of substrates may be produced or polymer microcastingtechniques where substrates with microscale and nanoscale features arepolymerized within a microfabricated mold.

The microfluidic liquid junction structure 102 may be one planar elementor a laminate of multiple planar elements. The planar elements may beattached to each other by a variety of means, including thermal bonding,adhesives, or in the case of glass and some plastics, direct fusion byheating to the melting point. The additional planar elements mayconstitute all or part of the array structure, or a rigid supportelement for the array structure element, or such additional layers mayinclude other microfluidic components that integrate into themicrofluidic liquid junction structure to provide increased performanceor additional features. Such additional elements might include microscale sensors and sensing elements that measure parameters such aspressure, flow rate, temperature, electrical resistance,oxidation-reduction (redox) potential, conductivity, and pH. Thesesensors could be utilized to provide feedback concerning the performanceof the potentiometric reference electrode 100 and the microfluidicliquid junction structure 102. Such feedback could be utilized bymonitor instrumentation for preventative diagnostics of the referenceelectrode's 100 performance. Such diagnostics might include determiningthe need for recalibration and predicting and signaling the need forservice well before the reference electrode 100 fails in an on-lineindustrial application.

FIG. 5 depicts an illustrative diametric cross-sectional view of thearray 105 of a microfluidic liquid junction structure 102 that isfabricated as a single planar polymer element. The planar element has aspecific density (channels/cm²) of etched anisotropic nanochannels 105.In this embodiment of the present invention specific channel densitiesof generally anisotropic nanochannels were fabricated in 10 μm thicksheets of polycarbonate.

The first step in the fabrication process was to expose 10 μm thicksheets of polycarbonate to charged particles, mostly heavy ions, in anuclear reactor. These charged particles perforate the polymer sheetsand leave “sensitized tracks” in the polymer which are substantiallyanisotropic. By controlling the duration of the exposure to the chargedparticles, the density of tracks per square centimeter can be controlledto a high degree of reproducibility. These tracks were generally uniformin width and straight, or anisotropic, and transverse the polymer sheetin a direction generally 90° to the planar axis of the polymer sheet.The tracks in the polymer substrate were preferably etched. This enabledthe nanochannels to be selectively etched to channel diameters of 10 nmand larger. The etching process consisted of immersing the polycarbonatesheets in a strong alkaline solution of 6 M NaOH with 10% methanol byvolume. To obtain sheets with different channel cross-sectional widthsthe etch times were varied from 1 hour to 1 minute.

In a final step in the fabrication process, the polycarbonate sheetswere coated by dipping them into a bath of 0.5% polyvinylpyrrolidone(PVP) solution. The PVP coating is hydrophilic and it enhances the“wetability” of the polycarbonate sheets and nanochannels. To obtainpolycarbonate sheets with nanochannels of cross-sectional diameters ofless than 10 nm, the inside walls of the 10 nm nanochannels wereuniformily plated with gold until the nanochannels were reduced to across-sectional width of 5 nm. By these fabrication techniques,polycarbonate planar elements with a range of nanochannel arrays thatcontained combinations of ultrasmall nanochannel cross-sectional widthsand low nanochannel densities that were not available from commercialsources were obtained and then analyzed.

By design of the nanochannel array 105 density, a flowing microfluidicliquid junction (MLJ) structure 102 was fabricated such that it had thedesire number of flowing nanochannels 106 exposed to aperture 130 on oneside of the microfluidic flowing liquid junction structure 102, and thecorresponding number of flowing nanochannels 106 exposed to orifice 132on the other side of the microfluidic flowing liquid junction structure102.

FIG. 6 depicts steps in the fabrication of a flowing microfluidic liquidjunction (MLJ) structure 164 from multiple polymer, polyimide planarelements 160 and 162 that may be thermal bonded together into onestructure. The two polyimide planar elements can be bonded togetherusing various techniques including those of U.S. Pat. No. 5,525,405(Coverdall et al.) and U.S. Pat. No. 5,932,799 (Moles).

Anisotropic nanochannels may be fabricated into the polymer polyimideplanar element 162 in the same manner as with the polycarbonate planarelement previously described above. The polyimide planar element 162 isfabricated to have a specific density of anisotropic nanochannels. Thethicker planar element 160 may also be fabricated from polyimide into ahoneycomb structure containing relatively larger, micron scale,microchannels 166 with cross-sectional widths on the order of 5 μm to 25μm in this embodiment. This honeycomb structure of the polyimide planarelement 160 adds mechanical strength to the finished microfluidicflowing liquid junction structure 164 without unduly impeding the forceof the pressurized electrolyte through the nanochannels 106. Thepolyimide planar element can be fabricated into a micron scale honeycombstructure by well known photolithography and wet etch techniques such asthose reported in U.S. Pat. No. 5,807,406 (Brauker et al.). Due to therelatively regular geometry of the resultant structure the resultantnumber of active flowing nanochannels 106 may be calculated as thenumber of nanochannels 106 that face a microchannel 166.

FIG. 7 depicts a schematic cross-section of the resultant flowingmicrofluidic liquid junction structure 164 that is made from twopolyimide planar elements, 160 and 162, that have been thermal bondedinto one structural element. On the average, each of the microchannels166 is connected to a small array 168 with approximately the same numberof nanochannels 106. In operation, pressurized electrolyte 110 entersinto an array 169 of micronchannels 166 and exits through the manyconnected nanochannels 106. In this way pressurized electrolyte 110flows through an array 169 of many smaller arrays 168 of nanochannels106. This is a useful technique to build up relatively thick planarstructures that do not unduly impede the pressurized flow of electrolyteinto the nanochannels 106.

In an alternative embodiment of the invention, additional planarelements of the same or different materials can be bonded on top of themicrofluidic flowing liquid junction structure 164 for additionalfeatures and performance such as additional strengthening structures,valves, or sensing elements. Such fabrication techniques are well knownand are reviewed by Marc Madou in “Fundamentals of Microfabrication”,1997, CRC Press. Referring to FIG. 1, it can be seen that thismicrofluidic flowing liquid junction structure 164 can be sealed intothe exemplary reference electrode 100 by compression means 122. Byproper selection of the nanochannel density of planar element 162 andthe microchannel density of planar element 160, a microfluidic flowingliquid junction structure 164 can be fabricated such that it has anmicrochannel array 169 with the desire number of flowing microchannels166 exposed to aperture 130 and the corresponding, connectingnanochannel arrays 168 with the desired number of flowing nanochannels106 exposed to orifice 132.

FIG. 8 depicts a flowing microfluidic liquid junction (MLJ) structure170 that can be fabricated from a single planar element of silicone bymeans of anisotropic plasma etching techniques such as those reported inU.S. Pat. No. 5,501,893 (Laermer et al.). The microfluidic flowingliquid junction structure 170 has micron scale microchannels 176 etchedin one side of the structure and connecting nanochannels 106 etchedthrough the other side of the structure.

FIG. 9 depicts a schematic cross-section of the silicone flowingmicrofluidic liquid junction (MLJ) structure 170. The flowingmicrofluidic liquid junction structure 170 has an array 179 ofmicrochannels 176 on one side of the structure that connect to an array178 of nanochannels 106 on the other side of the structure. In thisexemplary embodiment the ratio of nanochannels 106 that connect to eachmicrochannel 176 is one to one. Anisotopic plasma etching can fabricatehigh aspect ratio features in silicone with ratios as high as 20:1.Accordingly, in this embodiment the microchannels 176 can be etched 5 μmwide and 75 μm deep from one side of the structure and the nanochannels106 can be etched 100 nm wide and up to 2 μm deep from the other side ofthe microfluidic flowing liquid junction structure 170.

Again, the nanochannel array 178 density and the microchannel array 179density may be selected such that, a microfluidic flowing liquidjunction structure 170 may be fabricated such that it has a microchannelarray 179 with the desired number of flowing microchannels 176 exposedto aperture 130 and the corresponding, connected nanochannel array 178with the desired number of flowing nanochannels 106 exposed to orifice132. Such a junction may be designed to exhibit certain characteristicssuitable to any use.

FIG. 10 depicts steps in the fabrication of a flowing microfluidicliquid junction (MLJ) structure 184 from multiple glass planar elements180 and 182 that can be thermal bonded or fused together into one planarstructure. Planar element 180 is a solid element of glass, such asCorning 0120 glass, that has a single, relatively large channel 186 inthe center. The channel 186 can be several microns to 1 mm in diameterand it can be fabricated with well known microfabrication techniques.The planar element 182 is a glass disk that has at its center an array188 region of nanochannels. This planar element 182 can be made bymethods reported in U.S. Pat. No. 5,264,722 (Tonucci et al.) for themanufacture of nanochannel glass rod. Nanochannel glass rod made by thismethod is essentially a fused bundle of anisotropic glass tubes thateach have a cross-sectional width of just a few nanometer to severalhundred nanometers. Furthermore, the nanochannel glass rod, alsofabricated from Corning 0120 glass, can be clad in non-porous glass sothat just the core of the resultant glass rod is made up of an array 188of nanochannels. A single planar cross section 182 of this rod can becut to use as the nanochannel array 188 of the present embodiment of thepresent invention. The width of the nanochannels and the number ofnanochannels can be precisely controlled by the fabrication methodsreported in U.S. Pat. No. 5,264,722 (Tonucci et al.). The length of thenanochannels in the array 188 length can be controlled by cutting across-section of the rod and grinding it to the desired thickness.

Where both glass planar layers, 180 and 182, are made from the sameglass, they may be fused together into a single flowing microfluidicliquid junction (MLJ) structure 184 by scientific glass blowingtechniques well known to those skilled in the art. Alternatively, theymay be thermally bonded by the techniques disclosed and reviewed by MarcMadou in “Fundamentals of Microfabrication”, 1997, CRC Press.

FIG. 11 depicts a schematic cross-section view of the glass flowingmicrofluidic liquid junction (MLJ) structure 184. The flowingmicrofluidic liquid junction structure 184 has a single large channel186 on one side and a corresponding, connecting array 188 ofnanochannels 106 on the other side. The planar element 180 lendsmechanical strength to the planar element 181 in this embodiment of thepresent invention once they are bonded or fused together into the singleplanar flowing microfluidic liquid junction structure 184.

As before, by design of the nanochannel array 188 density and the sizeof the single channel 186, a microfluidic flowing liquid junctionstructure 184 may be fabricated such that the single channel 186 alignswith the aperture 130 and the corresponding, connected nanochannel array188 with the desired number of flowing nanochannels 106 are exposed toorifice 132.

TABLE 2 Representative Operational Specifications Of FlowingMicrofluidic Liquid Junctions Electrolyte Linear Velocity Range: greaterthan approximately 0.1 cm/sec Preferable range: greater thanapproximately 1.0 cm/sec Electrolyte Volumetric Flow Rate Range: lessthan approximately 1500 μl/day (500 ml/yr) less than approximately 60μl/hr Preferable range: less than approximately 150 μl/day (50 ml/yr)less than approximately 6 μl/hr Electrical Resistance Range: less thanapproximately 1 megohm Preferable range: less than approximately 100kohm

Experimental and Theoretical Data Based Upon Experimental Data

Table 3 and 4, presented below, detail certain actual physical andpotentiometric characteristics, and estimated physical andpotentiometric characteristics based upon and extrapolated from theactual physical and potentiometric characteristics, of microfluidicflowing liquid junctions of the invention having various structuralcharacteristics.

Table 3 provides experimental test data for reference electrodes havingexemplary flowing microfluidic liquid junction (MLJ) structures withinthe scope of the present invention. Transient, static and stirringerrors were determined in standard pH 7 buffer solutions afterconsecutive exposures to the test solution. The potential was measuredagainst a pH-sensitive glass electrode. The exemplary MLJ structurematerial was obtained from Osmonics Laboratory Products (Westborough,Mass., USA). The Osmonics part number for the 30 nm nanochannel MLJmaterial, P/N KN3CP01300; the Osmonics part number for the 50 nmnanochannel MLJ material, P/N KN5CP01300. The BJC Model 9015, P/NC2451C-12A, with typical commercially available diffusion junctionreference electrode was obtained from Broadley-James Corp. (Irvine,Calif., USA)

Table 4 provides the estimated resistance, velocity and lifetime ofexemplary MLJ structures within the scope of the present invention.Table 4 was generated based on the actual, experimentally determineddata derived from a MLJ structure with 1,000 10-μm long, nanochannelshaving widths of approximately 70 nm (see bottom row), speciallyprepared as described herein.

TABLE 3 Microfluidic Flowing Liquid Junction Reference Electrode TestsComparative Reference Electrode Tests: Microfluidic Flowing LiquidJunctions vs. Conventional Non-Flowing Diffusion Junction Test ReferenceChannel Channel Array Flow Rate Velocity Transient Error Static ErrorStirring Error Solution Electrode Width Length Size Pressure (μL/hr)(cm/s) (mV) (mV) (mV) pH 4 Buffer MLJ Design 50 nm 6 μm 1,000,000 40psig 1910 6.4 0.2 <0.1 −0.2 0.1 <0.1 <0.1 MLJ Design 30 nm 6 μm1,000,000 40 psig 70 0.7 0.3 <0.1 <0.1 <0.1 <0.1 <0.1 MLJ Design 50 nm 6μm 1,000,000 10 psig — — 0.1 <0.1 0.1 <0.1 <0.1 <0.1 MLJ Design 30 nm 6μm 1,000,000 10 psig — — 0.2 <0.1 0.1 <0.1 <0.1 <0.1 MLJ Design 70 nm 10μm    1000 40 psig 1.8 13 0.1 0.2 0.1 <0.1 0.3 0.4 BJC Model 9015 gelelectrolyte with non- N/A N/A N/A 1.5 0.6 −2.6 −2.9 1.3 0.9 flowingdiffusion junction 0.1 M HCl MLJ Design 50 nm 6 μm 1,000,000 40 psig 7412.5 <0.1 <0.1 <0.1 <0.1 0.1 0.2 MLJ Design 30 nm 6 μm 1,000,000 40 psig193 1.8 <0.1 <0.1 <0.1 <0.1 0.2 0.3 MLJ Design 50 nm 6 μm 1,000,000 10psig 114 0.4 <0.1 <0.1 <0.1 <0.1 <0.1 0.1 MLJ Design 30 nm 6 μm1,000,000 10 psig 20.3 0.2 <0.1 <0.1 <0.1 <0.1 0.1 0.1 BJC Model 9015gel electrolyte with non- N/A N/A N/A 5.2 −4.1 −2.3 2.2 0.6 1.7 flowingdiffusion junction 0.1 mM HCl MLJ Design 50 nm 6 μm 1,000,000 40 psig583 2.0 <0.1 <0.1 <0.1 0.2 <0.1 0.2 MLJ Design 30 nm 6 μm 1,000,000 40psig 20 0.19 <0.1 0.2 <0.1 0.3 0.9 1.2 MLJ Design 50 nm 6 μm 1,000,00010 psig 95 0.32 0.1 0.3 <0.1 <0.1 0.1 0.4 MLJ Design 30 nm 6 μm1,000,000 10 psig 47 0.44 0.1 0.2 <0.1 <0.1 0.1 0.2 BJC Model 9015 gelelectrolyte with non- N/A N/A N/A −3.2 22.5 −2.7 1.4 10.5 12 flowingdiffusion junction 1 M Tris MLJ Design 50 nm 6 μm 1,000,000 10 psig 3741.3 −0.2 −0.2 1.4 −0.9 −0.2 0.1 Buffer MLJ Design 30 nm 6 μm 1,000,00010 psig 39 0.4 0.9 1.1 −0.2 1.8 0.3 0.2 BJC Model 9015 gel electrolytewith non- N/A N/A N/A 24 26 −4.5 −1.9 1.6 0.8 flowing diffusion junction

TABLE 4 Electrode Characteristics/Lifetime Estimates for VariousJunction Designs Electrode Lifetime Estimates for Selected MLJ Designs(Derived from Junction Linear Flow and Resistance Data) EstimatedChannel Total Est. Linear Est. Lifetime Dimensions Array Size ResistanceVelocity (yrs) ID Length (# of (kΩ) (cm/s) for 50 mL (nm) (μm) Channels)(kiloohms) (40 psig) of Electrolyte 10 6 1,000 1,910.83 0.44 4568.11 106 10,000 191.08 0.44 456.81 10 6 100,000 19.11 0.44 45.68 10 6 1,000,0001.91 0.44 45.7 30 6 1,000 212.31 3.98 56.40 30 6 10,000 21.23 3.98 5.6430 6 100,000 2.12 3.98 0.56 30 6 1,000,000 0.21 3.98 0.06 50 6 1,00076.43 11.05 7.31 50 6 10,000 7.64 11.05 0.73 50 6 100,000 0.76 11.050.07 50 6 1,000,000 0.08 11.05 0.01 70 10  1,000 64.99 13.00 3.17

Technical, Computational and Theoretical Analyses

Although the invention is not limited to any specific explanation oftheory to explain why or under what conditions it performs as describedherein, the following technical, computational and theoretical analysesare advanced to explain the invention.

The technical aspects of the microfluidic flowing liquid junction of theinvention are addressed. The theoretical and practical requirements of astable liquid junction are described, and the advantages of using amicrofluidic flowing liquid junction are described and presented.Calculations and references demonstrate that the inventive use ofmicrofluidic and nanopore technology lead to a stable liquid junctionpotential.

Potentiometric measurements are necessarily made using two electrodes.One electrode is the sensing electrode, which changes its potential withthe concentration, or activity, of the analyte, e.g., a_(i) in eq. (1).The other electrode is the reference electrode, which ideally generatesa constant half-cell potential, E_(ref), eq. (1). The potential at eachelectrode is characteristic of the physicochemical state of theelectrode system, for example, the potential depends on temperature,pressure and the chemical composition of the system. The potential ofthe reference half-cell remains constant by placing the electrode in aseparate compartment with its own electrolyte. The reference compartmenthas a conductive path to the sample solution. The arrangement of theelectrode, the reference electrolyte and the conductive path is known asthe reference electrode. See Midgley, K.; Torrance, K. PotentiometricWater Analysis, 2^(nd) ed.; John Wiley & Sons: New York, 1991; p 12. Theinterface between the reference electrode and the sample solution is theliquid junction, which contributes a potential, E_(junc.). The sum ofthe sensing and reference electrode potentials, and the liquid junctionpotential is the measured cell potential, E_(cell), eq. (1).$\begin{matrix}{E_{cell} = {\left( {E_{i}^{o} + {\frac{RT}{n\quad F}\log \quad a_{i}}} \right) + E_{{ref}.} + E_{{junc}.}}} & (1)\end{matrix}$

In order to determine the liquid junction potential accurately (seeBates, R. G. The Determination of pH; John Wiley and Sons: New York,1973), or to minimize it (see Horvai, G.; Bates, R. G. Anal. Lett. 1989,22, 1293), the overall composition of the sample must be known a priori.However, in most chemical analyses the desire is typically not toprecisely determine or even minimize the liquid junction potential, butrather that the potential remain substantially constant and unchangingso that a reliable calibration can be made. There is typically no needto determine the liquid junction potential, but there is a need that thepotential be substantially invariant from one test measurement toanother at a given temperature and pressure. See IUPAC, Quantities,Units and Symbols in Physical Chemistry; Mills, I. Ed.; Blackwell:Oxford, 1993; p 62. Accurate potentiometric measurements thus depend onthe constancy of the liquid junction potential. However, there is afundamental limitation with the accuracy in potentiometric measurementsdue to a number of theoretical and practical limitations including adrifting, non-constant liquid junction potential.

The performance of a reference electrode not only depends on thechemical properties of the electrode, but also on the physicalarrangement of the liquid junction. The four main physical criterion ofsubstantially invariant liquid junction include, see Midgley, D.;Torrance, K. Potentiometric Water Analysis, 2^(nd) ed.; John Wiley andSons: New York, 1991; p 46, (i) the junction structure should beconstant, (ii) stirring or streaming of the sample solution should notaffect the reference potential, (iii) particulate matter from the sampleshould not clog the junction, and (iv) solution from one sample shouldnot be retained in the junction and carried over to the next sample. Theaccuracy of any potentiometric measurement thus depends on the abilityof the liquid junction design to meet these requirements.

Currently commercially available reference electrodes use an assortmentof liquid junction structures and designs to protect the referenceelectrolyte from the sample. These materials include porous ceramic,porous Teflon, wood, asbestos, and various fibers. Designs with doublejunctions, glass-sleeves, and fused salts are also used. All thesematerials and designs are meant to keep the reference environmentconstant. However, even if the reference solution remains unchanged, theliquid junction can become contaminated with the sample solution. Thisinevitably alters the potential of the liquid junction, and requires theelectrochemical sensor to be recalibrated. A changing liquid junction istypically why an electrochemical sensor requires frequent recalibration.

The most stable, reproducible, and reliable reference electrode designsincorporate a flowing-liquid junction. See Covington, A. K.; Whalley, P.D.; Davison, W. Anal. Chim. Acta 1985, 169, 221; Illingworth, J. A.Biochem. J. 1981, 195, 259; Wu, Y. C.; Feng, D.; Koch, W. F. J. SolutionChem. 1989, 18, 641; Ito, S.; Kobayashi, F.; 1 Baba, K.; Asano, Y.;Wada, H. Talanta 1996, 43, 13 5; Peters, G. Anal. Chem. 1997, 69, 2362;Lvov, S. N.; Zhou, X. Y.; Macdonald, D. D. J. Electroanal. Chem. 1999,463, 146; Brezinski, D. P. The Analyst 1983, 108, 425. The constant flowof reference electrolyte through the liquid junction helps it maintain aconstant composition by the continual renewal of fresh electrolyte. Thedisadvantage of using such an electrode is that it requires considerablemaintenance because the reference cell must be frequently refilled withelectrolyte. For this reason, flowing junctions are usually onlysuitable for the laboratory environment. Another problem of a typicalflowing-reference electrode is that if the sample is at a pressurehigher than the reference reservoir, the reference cell will readilybecome contaminated with the sample. Because of these disadvantages, inrecent years, the convenience and low maintenance of diffusion-stylejunctions has replaced the flowing-liquid junction in industrialapplication.

A superior flowing-liquid junction has been developed by combiningmicrofluidic materials and nanomaterials. The electrolyte has acontinual flow of small, manageable volumes of electrolyte through thejunction with a linear velocity sufficient to eliminate contamination ofthe junction and/or contamination of the reference electrolyte. Themicrofluidic flowing liquid junction provides the superior stability andperformance of a flowing liquid junction yet remain maintenance-free forextended periods of time, including a week, two weeks, a month, sixmonths, a year, or two years.

When miniaturizing chemical and physical processes, as in microfluidics,scaling laws must be considered. In addition, modeling fluid mechanicsrequires that correct assumptions as to the type of flow be made.Microfluidics typically have very low Reynolds numbers, Re<1, see Madou,M. Fundamentals of Microfabrication; CRC Press: New York, 1997; p 429.where viscous forces dominate. A consequence of viscous flow is thateach microscopic fluid element follows a fixed path or streamline. Anysubsequent fluid element, starting at the same point, will follow thesame streamline along its entire course. See Giddings, J. C. UnifiedSeparation Science; John Wiley and Sons: New York, 1991; pp 58-63. Sucha flow pattern creates a reproducible, non-varying, and predictablestructure, like that desired in a flowing-liquid junction. Tocharacterize the flow through a liquid junction the velocity profilesmust be determined.

To determine the velocity profile through a microchannel or nanochannel,all of the external forces acting on the fluid are to be balanced.First, the Newtonian acceleration (or inertial) forces are significantfor only a brief moment before steady flow is achieved in very smallchannels, see Giddings, J. C. Unified Separation Science; John Wiley andSons: New York, 1991; pp 58-63, and can be neglected. Second, all of thefluidic elements under consideration terminate as a sudden expansion.This implies that the kinetic energy of the fluid is not transferredfrom one element to the next. See Gravesen, P.; Branebjerg, J.; Jensen,O. S. J. Micromech. Microeng. 1993, 3, 168. Third, in very smallchannels gravitational forces may be neglected since the pressurerequired to induce steady flow is typically much larger than thegravitational force, i.e., Δp>>ρgh. See Giddings, J. C. UnifiedSeparation Science; John Wiley and Sons: New York, 1991; pp 58-63. Byneglecting acceleration, kinetic, and gravitational forces we need onlybalance the pressure acting against the viscous forces in order todetermine the velocity profile through a microchannel. Flow through verysmall channels is described by the Hagen-Poiseuille equation, eq. (2).The flux, Q (L/s), or the rate of flow through a cross-sectional area ofa channel is a function of the channel dimensions, the differentialpressure, and the properties of the solution. $\begin{matrix}{Q = \frac{{\pi\Delta}\quad p\quad r_{o}^{4}}{8\quad L\quad \eta}} & (2)\end{matrix}$

In eq. (2) Δp is the pressure differential at the two ends of thechannel, r_(o) and L are the radius and length of the channel,respectively, and η is the solution viscosity. (All of the calculationsin this proposal have assumed that the viscosity of the electrolyte isequal to 1.0 cp.) See All pure aqueous KCl solutions have a viscositybetween 0.9 and 1.1 cp. Hai-lang, Z.; Shi-Jun, H. J. Chem. Eng. Data1996, 41, 516. Examination of eq. (2) indicates that Q∝r_(O) ⁴, thus,simply constricting the cross section of a channel will greatly diminishthe flow through it. However, decreasing the cross-sectional area of achannel increases the electrolytic resistance. The conductance through acylindrical channel can be calculated by using eq. (3). $\begin{matrix}{G = {\frac{1}{R} = \frac{\lambda \quad C\quad \pi \quad r_{o}^{2}}{L}}} & (3)\end{matrix}$

The electrolytic resistance of the channel is taken as the reciprocal ofthe cell conductance, G. λ is the electrolyte conductance, C is theelectrolyte concentration, A and L are the cross-sectional area andlength of the channel, respectively. λ for a 4.0 M KCl solution is ˜10⁻²m² S mol⁻¹. See Handbook of Chemistry and Physics, 71^(st) ed.; Lide, D.R., Ed.; CRC Press: Ann Arbor, 1990. To minimize the electrolyteflow-rate and the resistance by simply reducing the size of a singlechannel is impractical, since the electrolytic resistance rapidlybecomes too high when the channel radius<˜1 μm. For example, thecalculated electrolytic resistance, using eq. (3), of a 1-mm longchannel with a 1-μm radius containing 4.0 M KCl is ˜8 MΩ. Thisresistance is too high for any realistic consideration, sinceelectrolytic resistance greater than approximately 500 kΩ is outside thecapabilities of typical commercial instrumentation. Fortunately, flowdecreases as the fourth power of the radius while resistance increasesas the square of the radius. Decreasing channel cross section butincreasing the number of channels is a practical way to reduce theelectrolytic resistance while maintaining the desired low flow.

Preferred embodiments of the present invention use an array ofnanochannels as a liquid junction structure to minimize both the flowrate and electrolytic resistance. For example, while a singlenanochannel with a 5-nm radius, and 6-μm long (see Nishizawa, M,; Menon,V. P.; Martin, C. R. Science 1995, 268, 700) has an electrolyticresistance of approximately 1000 MΩ in 4.0 M KCl (eq. (3)), and forexample, an array of 10⁵ nanochannels will have a resistance<100 kΩ.

Calculations thus far show that a microfluidic flowing liquid junctioncan provide the desired flow control and electrolytic conductivity toachieve a commercial product. Next, the electrolyte velocity needed tominimize the back diffusion of a sample into the liquid junction iscalculated. The average solution velocity through a single nanochannelcan be calculated by dividing the flux, Q, eq. (2), by thecross-sectional area of the nanochannel. $\begin{matrix}{v = \frac{Q}{\pi \quad r_{o}^{2}}} & (4)\end{matrix}$

Using eqs. (2) and (4), the flux through a nanochannel array and theaverage velocity (v) through a single nanochannel are plotted in FIG. 1as a function of the nanochannel radius. The calculations assume asteady pressure difference of 40 psi. The flux is plotted as the sensorlife assuming a 50-ml reservoir of electrolyte. The array contains 10⁵nanochannels and is 6 μm long. A 50 mL reservoir will be sufficient forcontinuous operation of a year or more for nanochannel radii less thanapproximately 30 nm. The radii of the nanochannels or microtubes mayalso have radii of less than approximately 20 nm, less thanapproximately 40 nm, less than approximately 50 nm, or less thanapproximately 60 nm. By increasing the volume of the reservoir, or bydecreasing the number or density of the nanochannels, the lifetime of asensor can be adjusted as needed, as will be appreciated by those ofordinary skill in the art.

An order of magnitude estimate of the electrolyte velocity needed todiminish diffusion of the sample into the liquid junction is calculated.A hydrodynamic model is used to model the convective-diffusion transportthrough a nanochannel. This model neglects electrostatic interactionsand migrational effects. Diffusion of the sample into the liquidjunction is described by Fick's first law, N_(D)=−D∇C, and theconvective flux is N_(v)=Cv. The sum of the diffusional and convectivefluxes is the total flux, eq. (5). $\begin{matrix}{N = {{{- D}\quad \frac{C}{x}} + {Cv}}} & (5)\end{matrix}$

In eq. (5) C is the concentration of the sample at position x in thechannel. ν is the convective velocity of the sample, and is approximatedas the average solution velocity through the channel. Integration of thecontinuity equation, Δ·N=0, with boundary conditions, C=C_(O) at x=l andC=0 at x=0, where C_(O) is the initial concentration of the sample, andl is the length of the nanochannel, yields the concentration profile forconvective-diffusion through a nanochannel. $\begin{matrix}{\frac{C}{C_{o}} = \frac{{\exp \quad \left( \frac{vx}{D} \right)} - 1}{{\exp \quad \left( \frac{vl}{D} \right)} - 1}} & (6)\end{matrix}$

The concentration profiles in the liquid junction are plotted as afunction of velocity in FIG. 13, using eq. (7). Apparently, solutionvelocities>˜0.1 cm/s should be sufficient to exclude the sample fromdiffusing into the reference reservoir. FIG. 12 shows a solutionvelocity of ˜0.1 cm/s can be generated for nanochannel radii>˜10 nm witha differential pressure of 40 psi. According to these calculations,nanochannels with radii between 10 to 40 nm will yield a constant,non-varying liquid junction that is low in resistance, and is operativefor at least one year.

Nanochannel arrays thus are shown theoretically to provide the idealapproach to solving the liquid junction problem.

Preferred Laboratory System Embodying the Invention

A system according to a preferred embodiment of the invention wasassembled. This system was used to test electrodes at controlledtemperatures, pressures and agitation rates. The system consists of a 50mL pressure cell, which can handle pressures as high as 45 psig asequipped. The laboratory test system mimics the different, sometimesharsh environments to which sensors may routinely be exposed inindustrial or field applications. The cell is exposed to temperaturesfor example, within 0.1° C., in a precision temperature bath. Amechanical stirrer provides adequate aeration and mixing of the testsolution. All of the instrumentation is linked to a computer for dataacquisition and archiving of the experimental measurements.

Theoretical Aspects of the Preparation and Characterization of theNanochannel Array

The Au nanochannel arrays that were used as the liquid junctionstructure were prepared via a general approach for preparingnanomaterials called “template synthesis.” See Hulteen, J. C.; Martin,C. R. J. Mater. Chem. 1997, 7, 1075. The template method entails thesynthesis of a desired material within the channels of a microporousmembrane. The membranes employed have cylindrical channels withmonodisperse diameters that run the complete thickness of the membrane.Corresponding cylindrical nanostructures of the desired material areobtained within the channels.

A commercially available microporous polycarbonate filtration membranemay be used as the template to prepare the nanochannel arrays. Thismembrane contains monodisperse and cylindrical pores. An electronicplating procedure is used to deposit Au nanochannels within these pores.See Nishizawa, M,; Menon, V. P.; Martin, C. R. Science 1995, 268, 700;Hulteen, J. C.; Martin, C. R. J. Mater. Chem. 1997, 7, 1075; Hulteen, J.C.; Martin, C. R. J. Am. Chem. Soc. 1998, 26, 6603; Menon, V. P.;Martin, C. R. Anal. Chem. 1995, 67, 1920. This Au plating procedure iswell known in the art.

The template membrane may be first rinsed in methanol and then immersedin a 0.025 M SnCl₂ and 0.07 M in trifluoroacetic acid solution. Thisresults in “sensitization” of the membrane, typically meaning theadsorption of Sn(II) to the channel walls and membrane surfaces. Thesensitized membrane is then immersed into an aqueous solution ofammoniacal AgNO₃. This causes the following surface redox reaction,

2 Ag⁺+Sn(II)→2 Ag^(o)+Sn(IV)  (7)

and the channel walls and membrane phases become coated with nanoscopicAg particles. These particles act as the initial catalyst forelectroless Au deposition. Finally, the membrane may be placed in a goldplating bath, which contains 0.5 mL of a commercially-available goldplating solution, 0.127 M Na₂SO₃, 0.625 M formaldehyde and 0.025 MNaHCO₃. The solution may be adjusted to pH 10 by dropwise addition of0.5 M H₂SO₄. The temperature of this plating bath is typicallymaintained at 5° C. The inside diameter of the Au nanochannels depositedwithin the pores of the array is adjusted by varying the plating time,which typically refers to the immersion time in the Au plating bath.

This procedure is optionally used to prepare arrays containing Aunanochannels with inside diameters of molecular dimensions (<1 nm)., SeeNishizawa, M,; Menon, V. P.; Martin, C. R. Science 1995, 268, 700;Hulteen, J. C.; Martin, C. R. J. Mater. Chem. 1997, 7, 1075; Hulteen, J.C.; Martin, C. R. J. Am. Chem. Soc. 1998, 26, 6603; Menon, V. P.;Martin, C. R. Anal. Chem. 1995, 67, 1920; Petzny, W. J.; Quinn, J. A.Science 1969, 166, 751. Ion-transport in these arrays has been studied,see Nishizawa, M,; Menon, V. P.; Martin, C. R. Science 1995, 268, 700.The resulting nanochannels are ion permselective and may be reversiblyswitched between anion-transporting and cation-transporting states.

The inside diameters of the Au nanochannels may be readily approximatedby measuring the flux of H₂ gas across the nanochannel array. SeePetzny, W. J.; Quinn, J. A. Science 1969, 166, 751. See also Liu, C.,Texas A&M University, College Station; 1991. The nanochannel samples arethen placed in a vacuum oven for at least 12 hours prior to making theflux measurements, to remove traces of water or other volatile speciesabsorbed in the nanochannels. Reproducible values of flux are bestobtained when nanochannels are pretreated in this manner. Thenanochannel array may then be placed in the gas-permeation cell, and theupper and lower half-cells evacuated. The upper half is pressurized to20 psig with H₂, and the pressure-time transient associated with leakageof H₂ through the nanochannels into the lower half-cell was measured.This is converted to the flux of gas, from which the average nanochanneldiameter may be approximated. Assuming gas-transport through ananochannel array occurs via Knudsen diffusion, the flux of gas, Q_(gas)(moles cm⁻² s⁻¹), is related to the pore density, n (pores cm⁻²), thepore diameter, d (cm), and the membrane thickness, L (cm) using eq. (8).$\begin{matrix}{{({gas})\quad Q} = \frac{8\quad \pi \quad {nd}^{3}\Delta \quad p}{3{MRTL}}} & (8)\end{matrix}$

Δp is the pressure difference across the membrane (dynes cm⁻²), M is themolecular weight of the gas, R is the gas constant (erg K⁻¹ mol⁻¹), andT is the temperature (K). In our experiment, we know all of theparameters in eq. (8), except d.

A variety of nanochannel arrays of various sizes and materials may beconstructed and used. These include different radii for example, (10,20, 30 and 40 nm), and substrate materials, for example, (polycarbonateand polyester), and two Au surfaces. The inside diameter of thenanochannels may be varied by the plating time, which have beencharacterized for precise nanochannel dimensions. Au nanochannels and Aunanochannels with an adsorbed propanethiol monolayer are preferred.Chloride ions readily adsorb on gold surfaces, thus, in 4.0 M KClreference solutions the Au nanochannels will have a net negative charge.However, the nanochannels pretreated with propanethiol have an inert,uncharged monolayer that prevents chloride ions from adsorbing.

Alternatively, addition of a propanthiol monolayer is accomplished byimmersing the array into an ethanol solution containing the thiol. Thissmall thiol molecule does not appreciably change the nanochannel insidediameter when the diameter>˜5 nm. For this reason, there is no need toredetermine the nanochannel inside diameter after chemisorption of thethiol. In addition, the propanethiol-modified Au nanochannels remainwater “wetable” after addition of the thiol. See Nishizawa, M,; Menon,V. P.; Martin, C. R. Science 1995, 268, 700.

The various articles of the scientific and/or medical literature, andthe U.S. and international and/or foreign patents and patentapplications cited herein are hereby incorporated by reference to theextent permitted by law. To the extent that each is incorporated byreference herein, each constitutes a part of the disclosure of thisspecification. Furthermore, specific embodiments, working examples, andprophetic examples of the invention have been described in detail toillustrate the broad applicability and principles underlying theinvention, such as the use of microfluidic flowing liquid junction aspart of a reference electrode or as part of a combination electrode, andthe various methods of manufacturing and/or using the microfluidicflowing liquid junction, or of manufacturing and/or using a referenceelectrode or a combination electrode comprising a microfluidic flowingliquid junction. Notwithstanding these specific embodiments, workingexamples, and prophetic examples, it will be understood by those ofskill in the art that the invention may be embodied otherwise withoutdeparting from such broad applicability and principles.

What is claimed is:
 1. A flowing junction reference electrode comprising: a microfluidic liquid junction member situated between a pressurized reference electrolyte solution and a sample solution, the microfluidic liquid junction member having an array of fewer than approximately 10⁵ discrete nanochannels.
 2. The electrode of claim 1, wherein the number of nanochannels is greater than approximately
 10. 3. The electrode of claim 2, wherein the number of nanochannels is less than approximately 10³.
 4. The electrode of claim 2, wherein the number of nanochannels is less than approximately 10⁴.
 5. The electrode of claim 2, wherein the number of nanochannels is less than approximately
 100. 6. The electrode of claim 1, wherein the nanochannels are substantially straight and substantially parallel to one another.
 7. The electrode of claim 1, wherein the width of any nanochannel in the array of nanochannels is substantially equal to the width of any other nanochannels in the array of nanochannels.
 8. The electrode of claim 1, wherein the nanochannels have widths of greater than approximately 1 nanometer and less than approximately 900 nanometers.
 9. The electrode of claim 1, wherein the nanochannels have widths of greater than approximately 10 nanometers and less than approximately 500 nanometers.
 10. The electrode of claim 1, wherein the nanochannels are coated.
 11. The electrode of claim 1 wherein the junction member comprises a polymer.
 12. The electrode of claim 11 wherein the polymer is selected from the group consisting of polycarbonate and polyimide.
 13. The electrode of claim 1 wherein the junction member comprises a material selected from the group consisting of silicon, glass, and ceramic.
 14. The electrode of claim 13, wherein the junction member comprises glass.
 15. The electrode of claim 1, further comprising means for maintaining positive linear flow of the reference electrolyte solution through the array of nanochannels and into the sample solution.
 16. The electrode of claim 15 wherein the means for maintaining positive linear flow of the reference electrolyte solution through the array of nanochannels and into the sample solution is selected from the group consisting a pressurized collapsible bladder, an electro-osmotic pump, a mechanical pump, a piezo-electric pump, and a electro-hydrodynamic pump.
 17. The electrode of claim 1, wherein the resistance across the junction member is less than approximately 1 megohm.
 18. The electrode of claim 1, wherein the number of nanochannels is greater than approximately
 100. 19. The electrode of claim 18, wherein the number of nanochannels is less than approximately 10⁴.
 20. The electrode of claim 18, wherein the number of nanochannels is less than approximately 10³.
 21. The electrode of claim 1, wherein the number of nanochannels is greater than approximately
 1000. 22. The electrode of claim 21, wherein the number of nanochannels is less than approximately 10⁴.
 23. The electrode of claim 1, further comprising means for maintaining positive linear flow of the reference electrolyte solution through the array of nanochannels and into the sample solution at a linear velocity greater than about 0.1 centimeter per second.
 24. The electrode of claim 23 wherein the means for maintaining positive linear flow of the reference electrolyte solution through the array of nanochannels and into the sample solution at a linear velocity greater than about 0.1 centimeter per second is selected from the group consisting a pressurized collapsible bladder, an electro-osmotic pump, a mechanical pump, a piezo-electric pump, and a electro-hydrodynamic pump.
 25. A combination electrode comprising: a flowing liquid junction reference electrode comprising a microfluidic junction member situated between a reference electrolyte solution and a sample solution, the microfluidic junction member having an array of fewer than approximately 10⁵ discrete nanochannels; and a sensing electrode.
 26. The combination electrode of claim 25, wherein the sensing electrode is selected from the group consisting of pH electrodes, other ion-selective electrodes, and redox electrodes. 